Protocol 39821: Probe Assay - Bicra<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr7:15976839-15976948 110bp ACAGTTTCCCTTCTGCCAAG GTGACCCTGACATCAAACTGG

Mut = 100 bp

Wt = 110 bp

Fam = Mut

Hex = Wt

 

Sequence

MUT Sequence (junction in uppercase):

aaaagagcccagttcccaagtgcagtgcccagaacatccacccacacacacccatttcccatggctggccattCCatctccagtttgatgtcagggtcactgtttcttcagcttgtcaggtgtggtgggga

This mutation is a 3648 bp deletion beginning at Chromosome 7 position 15,976,865 bp and ending after 15,980,512 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
54693 GTG ACC CTG ACA TCA AAC TGG Common A
54694 ACA GTT TCC CTT CTG CCA AG Wild type Forward A
54695 AAA AGA GCC CAG TTC CCA AG Mutant Forward A
54696 Fluorophore-1 ATT GGC AGG CTG CCG AC Quencher-1 WT Probe
54697 Fluorophore-2 CCA TGG CTG GCC ATT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
54693 0.40 uM
54694 0.40 uM
54695 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.